跳到主要內容

臺灣博碩士論文加值系統

(44.200.175.255) 您好!臺灣時間:2022/08/11 12:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:簡嘉慧
研究生(外文):Chia-Hui Chien
論文名稱:大腸桿菌天冬胺酸激酶III之定點突變及特性分析
論文名稱(外文):Site-directed mutagenesis and characterization of Aspartokinase III in E. coli
指導教授:許宗雄許宗雄引用關係
指導教授(外文):Tzong-Hsiung Hseu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:77
中文關鍵詞:天冬胺酸激酶III定點突變離胺酸
外文關鍵詞:Aspartokinase IIISite-directed mutagenesisLysine
相關次數:
  • 被引用被引用:0
  • 點閱點閱:208
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
使用定點突變的方式,將T352分別以聚合酶連鎖反應突變為T352A、T352D、T352G、T352N、T352V及T352P等六個突變株,再轉形至DH5α內表現,粗酵素液經streptomysine sulfate及ammonium sulfate分別沉澱大量核酸及含天冬胺酸激酶的部分蛋白,再以Superdex 75及Mono-Q column進行純化,此五種突變株及野生種天冬胺酸激酶的最後的純化倍數約在1.7~2.1之間,而活性回收則在13~30 %之間。最後分析其酵素動力學性質,以了解與受質之結合機制及關係。在離胺酸敏感性試驗結果顯示,突變為非極性胺基酸Ala及Val及Pro對離胺酸較其他突變株不敏感,而其中以Asn對離胺酸最為敏感。但有趣的是Ala、Val以及Pro分別偏好三種完全不同的二級結構,依序是α-helices、β-sheet及turns,所以有可能二級結構的改變並非是影響對離胺酸敏感與否的決定性因子,但這都只是初步推測,因為事實上所有突變株在352位置附近的真實結構為何,並無法確實得知。對照動力學常數結果,在關於對substrate binding的影響裡,T352A及T352V的 皆比wild-type高,似乎亦可說明,當被hydrophobic group (alanine, valine)的胺基酸取代後,會降低對aspartic acid binding affinity,且T352A及T352V的 也較wild-type高,顯示T352A及T352V比wild-type在形成E-ASP-ATP之三元複合體時較容易解離。在 常數方面,T352D及T352N分別為10.21及17.32,是五種突變株中最高的,而且也較其他的突變株對離胺酸敏感。而且在 方面,T352D為9.76 mM及T352G為7.3 mM是較高的兩株,同時T352G也較對離胺酸敏感。

In lysine biosynthesis of Escherichia coli, aspartokinase III takes part and plays a key role in the phosphorylation of aspartic acid. Aspartokinase III is a allosteric enzyme, and feedback-inhibited by lysine. The kinetic and regulatory properties of lysine-sensitive AK III are well characterized and lysine inhibition of the AK III enzyme is cooperative and noncompetitive with aspartic acid. Interactions between subunits have also been demonstrated to exhibit a ligand induced association - dissociation behavior.
Previously, a mutant lysC gene has been cloned by spontaneous mutation of a recombinant plasmid harboring a wild-type lysC gene. Nucleotide sequence determinations of the mutant lysC gene show only A to C substitution, and Thr to Pro at position 352 (T352P). After kinetic and binding studying, it shows that the point mutation implicates the alleviation of binding affinity for substrate and loss of the binding site of allosteric inhibitors, lysine and leucine.
In order to figure out the possible effect of the position 352 in AK III, the methodology of site-directed mutagenesis was taken, and the residue 352 was changed from Thr to Ala, Asp, Gly, Asn and Val. Then the changed target amino acid residue at 352 position of pUC19AK3 plasmids were transformed to Escherichia coli DH5α and expression. To isolate the five mutant AK III protein, Superdex 75 gel filtration column and Mono-Q anion exchange column were used.
In steady state initial velocity study, kinetics was done by verifying the concentration of one substrate at a fixed concentration of the other. Therefore the initial velocity could be calculated and the primary plots (double reciprocal plot) and secondary plot were constructed.
The lysine inhibition results reveal that when position 352 was substituted by hydrophobic amino acid residues, like Ala and Val, the residual activity of the two mutant AK III are much higher than those substituted by hydrophilic amino acid, like Asp, Gly and Asn. And the and values are agree with the result.
The of T352D(10.21 ) and T352N(17.32 ) are higher than other mutants, and they are also more sensitive to lysine. Comparing values, T352D and T352 G are higher and also more sensitive to lysine than T352A and T352V.

目錄 1
摘要 5
Abstract 6
謝辭 7
圖表目錄 3
第一章 序論 8
第二章 材料與方法 12
藥品與材料 12
菌種來源 13
小量質體DNA的製備 14
定位突變 (Site-directed mutagenesis) 15
洋菜膠電泳 16
製備competent cells 17
菌體轉形 17
突變殖株的篩選 17
酵素活性的測定 18
培養條件 18
酵素的純化 19
聚丙醯胺凝膠電泳分析(SDS-PAGE) 21
蛋白質濃度測定 21
酵素動力學性質的測定 22
酵素動力學數據分析 23
第三章 結果 24
定點突變 24
突變殖株的篩選 24
酵素的純化 25
酵素動力學研究 25
離胺酸與白胺酸的抑制研究 26
第四章 討論 27
參考文獻 31
圖表附錄 33

1.J. C. Patte, (1983) in Amino acid Biosynthesis and Genetic Regulation Somerville and Hermann (Eds).
2.J. Theze, D. Margarita, G. N. Cohen, F. Borne and J. C. Patte, J. Bacteriol. 117 (1974), 133-143.
3. C. Yanofaky, Nature 289 (1977) 751-758
4. H. H. Liao and T. H. Hseu, FEMS Microbiol. Lett. 168 (1998) 31-36
5.Richaud, C., Mazat, J. P., Gros, C., and Patte, J. C. (1973), Eur. J. Biochem. 40, 619-629
6.Richaud, C., Mazat, J. P., Felenbbock, B., and Patte, J. C. (1974), Eur. J. Biochem. 48, 147-156
7.Patte, J. C., Loviny, T., and Cohen, G. N. (1965), Biochim. Biophys. Acta 99, 523-530
8.Stadtman, E. R., Cohen, G. N., Lebras, G., and de Robichon Szulmajester, H. (1961), J. Biol. Chem. 236, 2033-2038
9.Truffa-Bachi, P., and Cohen, G. N. (1966), Biochim. Biophys. Acta 113, 531-541
10.Wampler, D. E., and Westhead, E. W. Two Aspartokinase from Escherichia coli. Nature of the Inhibition and Molecular Changes Accompanying Reversible Inactivation, (1968), Biochemistry 7, 1661
11.Monod, J., Wyman, J. & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118
12.K.J. Huang, and T.H. Hseu, Proceed. Natl. Sci. Council. ROC. 17 (1993) 91-97
13.C.C. Chen, (1991) MS Thesis, National Tsing Hua University.
14.M.K. Lin, (1997) MS Thesis, National Tsing Hua University.
15.Chou, P. Y. and Fasman, G. D. (1978) Adv. Enzymol. 47:45-148
16.Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular cloning : A laboratory Manual. Cold Spring Harbor Laboratory, Cold spring Harbor, NY. 1989
17.Fisher, C. L., and Pei, G. K(1997) Biotechniques 23, 570-574
18.Inoue, H., Nojima, H., and Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990; 96, 23-28
19.Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227, 680-685
20.Tikhonov, V. N. and Mustafin, I. S. (1965) Zh. Anal. Khim. 20, 390
21.Mazonski, T. et al. (1963) Zeszyty. Nauk. Politech. Slask. Chem. 13, 63
22.Smith, P. k. et al. (1985) Anal. Biochem. 150, 76
23.Yuri Ogawa-Miyata, Hiroyuri Kojima, and Konosuke Sano (2001) Biosci. Biotechnol. Biochem, 65 (5), 1149-1154

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top